Bispecific anti-cd19x anti-cd16 antibodies and uses thereof

ABSTRACT

Described are multivalent multimeric antibodies comprising at least two binding sites specific for the human B cell marker CD19 and human Fc γ  receptor III (CD16). Also described are polynucleotides encoding said antibodies as well as vectors comprising said polynucleotides, host cells transformed therewith and their use in the production of said antibodies. Finally, compositions are described comprising any of above mentioned antibodies, polynucleotides or vectors. The pharmaceutical compositions are useful for immunotherapy, preferably against B cell malignancies such as non-Hodgkin&#39;s lymphoma.

The present invention relates to multivalent multimeric antibodiescomprising at least two binding sites specific for the human B cellmarker CD19 and human Fc_(γ) receptor III (CD16). The present inventionalso relates to polynucleotides encoding said antibodies as well asvectors comprising said polynucleotides, host cells transformedtherewith and their use in the production of said antibodies. Finally,the present invention relates to compositions, preferably pharmaceuticaland diagnostic compositions, comprising any of above mentionedpolynucleotides, antibodies or vectors. The pharmaceutical compositionsare useful for immunotherapy, preferably against B cell malignanciessuch as non-Hodgkin's lymphoma.

Non-Hodgkin's lymphoma (NHL) encompasses a heterogeneous group ofhematological malignancies of B and T cell origin occurring in blood,lymph nodes and bone marrow, which commonly disseminate throughout thebody. NHL is one of the few malignancies that have increased infrequency more than the increase in population, with approximately53,000 new cases occurring annually in the United States. The mostcommon forms of NHL are derived from the B cell lineage. While NHL canbe treated with reasonable success at early and intermediate stages, theresults of conventional chemotherapy and radiation in advanced stagesremain disappointing. This holds particularly true for the prevalentlow-grade lymphomas. A fairly large number of patients relapse and mostremissions cannot be extended beyond the minimal residual disease. Thisdiscouraging situation has stimulated the search for alternativetherapeutic strategies, such as activation of host immune mechanismsusing bispecific antibodies (BsAbs) (van Spriel et al., Immunol. Today21 (2000), 391-397). The BsAb makes a bridge between the tumor cell andthe immune effector cell followed by triggering the cytotoxic responsesthat include perforin and granzyme release, Fas-mediated apoptosis andcytokine production. Since NHLs typically express one or more B cellmarkers, e.g. CD19 or CD20, these markers can be used to redirecteffector cells towards malignant B cells. Although normal B cells willbe also destroyed, they are repopulated from stem cells lacking thetargeted antigens. To mediate redirected lysis, a BsAb must bind atarget cell directly to a triggering molecule on the effector. Thebest-studied cytotoxic triggering receptors are multichain signalingcomplexes, such as T cell receptor (TCR)/CD3 complex on the T cell,Fc_(γ)RIIIa (CD16) on natural killer (NK) cell, Fc_(γ)RI (CD64) andFc_(γ)RI (CD89) expressed by monocytes, macrophages, and granulocytes.BsAbs directed to TCR/CD3 complex have the potential to target all Tcells, regardless of their natural MHC specificity. Thus far, differentforms of CD19×CD3 BsAb have been generated and used in a number of invitro and in vivo therapeutic studies. These BsAbs have mainly beenproduced using rodent hybrid hybridomas or by chemical cross-linking oftwo monoclonal antibodies. However, the human anti-murine antibody(HAMA) response and release of inflammatory cytokines are the majordrawbacks of BsAb derived from rodent antibodies in clinical use.Moreover, CD3-based immunotherapy requires additional stimulation of Tcell population via a signal delivered by a distinct co-receptor.Therefore, the thus far available BsAbs suffer from low T cellcytotoxicity and the need of costimulatory agents in order to displaysatisfactory biological activity.

Thus, the technical problem underlying the present invention was toprovide means suitable for therapy of B-cell malignancies that overcomethe disadvantages of the means of the prior art.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims. The present invention is basedon the observation that the generation of antibodies relying onretargeting of NK cells has a positive therapeutic effect, since, unlikeT cells, FcR-bearing cellular mediators of innate immunity as e.g. NKcells (and monocytes, macrophages and granulocytes) tend to exist inconstitutively activated states and do not need additional(pre-)stimulation.

Furthermore, monoclonal BsAbs used so far for therapy haveimmunoglobulin constant domains, which are responsible for undesiredimmune reactions (HAMA response). Thus, a preferred embodiment of theantibodies of the present invention are BsAb which only comprise thevariable immunoglobulin domains, so called F_(v) modules by means ofwhich undesired immune responses can be avoided. Furthermore, they havea stability that makes it usable for therapeutic uses. The F_(v) moduleis formed by association of the immunoglobulin heavy and light chainvariable domains, V_(H) and V_(L), respectively. The bispecificmolecule, so called bispecific diabody (BsDb), can be formed by thenoncovalent association of two single-chain fusion products, consistingof the V_(H) domain from one antibody connected by a short linker to theV_(L) domain of another antibody. Alternatively, recombinant BsAb,tandem diabody (Tandab) can be formed by homodimerization ofsingle-chain molecules comprising four antibody variable domains (V_(H)and V_(L)) of two different specificities, in an orientation preventingintramolecular F_(v) formation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Genetic Organization of Operon Encoding CD19×CD16 Bivalent BsDb(A) and Protein Model of BsDb (B)

His₆: six C-terminal histidine residues; L: short 10 amino acid peptidelinker SerAlaLysThrThrProLysLeuGlyGly connecting V_(H) and V_(L)domains; leader, bacterial leader sequence (e.g. PelB leader) forsecretion of recombinant product into periplasm; p/o: promoter/operator;rbs, ribosome binding site; Stop: stop codon (TAA); tag: C-terminalepitope for immunodetection; V_(H) and V_(L): variable regions of theheavy and light chains specific either to CD16 or to CD19.

FIG. 2: Genetic Organization of Operon Encoding CD19×CD16 TetravalentTandab (A) and Protein Model of Tandab (B)

His₆: six C-terminal histidine residues; L: short 10 amino acid peptidelinker SerAlaLysThrThrProLysLeuGlyGly connecting V_(H) and V_(L)domains; leader, bacterial leader sequence (e.g. PelB leader) forsecretion of recombinant product into periplasm; p/o: promoter/operator;rbs, ribosome binding site; SL: 12 amino acid peptide linkerArgAlaAspAlaAlaAlaAlaGlyGlyProGlySer between domains in the middle ofthe molecule; Stop: stop codon (TAA); tag: C-terminal epitope forimmunodetection; V_(H) and V_(L): variable regions of the heavy andlight chains specific either to CD16 or to CD19.

FIG. 3: Diagram of the Expression Plasmid pKID19×16

6×His: sequence encoding six C-terminal histidine residues; bla: gene ofbeta-lactamase responsible for ampicillin resistance; bp: base pairs;c-myc epitope: sequence coding for an epitope which is recognized by the9E10 antibody; ColE1: origin of the DNA replication; f1 IR: intergenicregion of bacteriophage f1; lac P/O: wild-type lac-operonpromoter/operator; Linker L: sequence which encodes the 10 amino acidpeptide SerAlaLysThrThrProLysLeuGlyGly connecting the V_(H) and V_(L)domains; PelB leader: signal peptide sequence of the bacterial pectatelyase; RBS: lacZ ribosome binding site; V_(H) and V_(L): sequence codingfor the variable region of the immunoglobulin heavy and light chain,respectively. Unique restriction sites are indicated.

FIG. 4: Diagram of the Expression Plasmid pSKID19×16

6×His: sequence encoding six C-terminal histidine residues; bla: gene ofbeta-lactamase responsible for ampicillin resistance; bp: base pairs;c-myc epitope: sequence coding for an epitope which is recognized by the9E10 antibody; hok-sok: plasmid stabilizing DNA locus; lacI: geneencoding lac-repressor; lac P/O: wild-type lac-operon promoter/operator;Linker L: sequence which encodes the 10 amino acid peptideSerAlaLysThrThrProLysLeuGlyGly connecting the V_(H) and V_(L) domains;M13 IR: intergenic region of bacteriophage M13; pBR322ori: origin of theDNA replication; PelB leader: signal peptide sequence of the bacterialpectate lyase; SD1: Shine-Dalgarno sequence (ribosome binding site)derived from E. coli lacZ gene (lacZ); SD2, SD2 and SD3: Shine-Dalgarnosequence for the strongly expressed gene 10 of bacteriophage T7 (T7g10);skp gene: gene encoding bacterial periplasmic factor Skp/OmpH; tHP:strong transcriptional terminator; tLPP: lipoprotein terminator oftranscription; V_(H) and V_(L): sequence coding for the variable regionof the immunoglobulin heavy and light chain, respectively. Uniquerestriction sites are indicated.

FIG. 5: Nucleotide and Deduced Amino Acid Sequences of the CD19×CD16BsDb in the Expression Plasmid pSKID19×16

6×His: sequence encoding six C-terminal histidine residues; CDR:complementarity determining region of the immunoglobulin heavy (H) orlight (L) chain; c-myc epitope: sequence coding for an epitope which isrecognized by the 9E10 antibody; lac P/O: wild-type lac-operonpromoter/operator; laczí: gene encoding the amino terminal peptide ofbeta-galactosidase; Linker L: sequence which encodes the 10 amino acidpeptide SerAlaLysThrThrProLysLeuGlyGly connecting the V_(H) and V_(L)domains; pelB leader: signal peptide sequence of the bacterial pectatelyase; SD1: Shine-Dalgarno sequence (ribosome binding site) derived fromE. coli lacZ gene (lacZ); SD2, SD2 and SD3: Shine-Dalgarno sequence forthe strongly expressed gene 10 of bacteriophage T7 (T7g10); V_(H) andV_(L): sequence coding for the variable region of the immunoglobulinheavy and light chain, respectively. The restriction sites used for geneassembly are indicated. The start and stop codons of translation areshown in bold.

FIG. 6: Analysis of Purified CD19×CD16 BsDb by Size ExclusionChromatography on a Calibrated Superdex 200 Column

The elution positions of molecular mass standards are indicated.

FIG. 7: Analysis of Purified CD19×CD16 BsDb by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Under ReducingConditions

Lane 1, M_(r) markers (kDa, M_(r) in thousands); Lane 2, CD19×CD16 BsDb.The gel was stained with Coomassie. Positions of hybrid V_(H)1⁶-V_(L)1⁹and V_(H)1⁹-V_(L)1⁶ scFvs are indicated.

FIG. 8: Lineweaver-Burk Analysis of Fluorescence Dependence on CD19×CD16Diabody Concentration as Determined by Flow Cytometry

Diabody binding to CD19⁺ JOK-1 cells and CD16⁺ 293-CD16 cells wasmeasured.

FIG. 9: Retention of CD19×CD16 BsDb on the Surface of CD19⁺ JOK-1 Cellsand CD16⁺ 293-CD16 Cells at 37° C.

Values are expressed as a percentage of initial mean fluorescenceintensity.

FIG. 10: Lysis of CD19⁺ Raji Cells by Human PBLs at DifferentEffector:Target (E:T) Ratios in Presence of CD19×CD16 BsDb atConcentration of 0.5, 1 and 5 mg/ml

FIG. 11: Lysis of CD19⁺ Raji cells by Human NK Cells at Different E:TRatios in Presence of CD19×CD16 BsDb at Concentration of 0.5, 1 and 5mg/ml.

FIG. 12: Treatment of Severe Combined Immunodeficiency (SCID) MiceBearing Human Burkitt's Lymphoma Xenografts

The mice received PBS (open squares), human PBLs alone (filled squares),or human PBLs followed 4 h later by the administration of CD19×CD16 BsDb(filled circles). Tumor size was measured every second day. Tumor growthcurves of individual animals till 30 day of experiment are presented.

FIG. 13: Survival of SCID Mice Bearing Human Burkitt's LymphomaXenografts

The mice received PBS (open squares), human PBLs alone (filled squares)or human PBLs followed 4 h later by the administration of CD19×CD16 BsDb(filled circles).

Accordingly, the present invention relates to a multivalent multimericantibody, characterized by the following features:

-   -   (a) it has at least two specificities;    -   (b) at least one antigen-binding domain is specific to human        CD19; and    -   (c) at least one antigen-binding domain is specific to human        CD16.

The antibody of the present invention is specific to both human CD19 onmalignant B cells and to human CD16 on cytotoxic NK cells. Such antibodyis capable of destroying CD19-positive tumor cells by recruitment ofhuman NK cells without any need for pre- and/or co-stimulation. This isin sharp contrast with any known bispecific molecules retargetingeffector cells to CD19-positive target cells, such as CD19×CD3 orCD19×CD28 BsAbs. The independence from pre- and/or co-stimulation ofeffector cell population may substantially contribute to the therapeuticeffect of such antibodies by augmentation of adverse side effects, suchas cytokine release syndrome.

The antibodies of the present invention can be prepared by methods knownto the person skilled in the art, e.g. by the following methods:

-   -   (a) Chemical coupling of antibodies or antibody fragments        specific to human CD19 and CD16, respectively, via        heterobifunctional linkers.    -   (b) Fusion of hybridoma cell lines which are already available        and which secrete monoclonal antibodies specific to human CD19        and CD16, respectively.    -   (c) Transfection of the immunoglobulin light and heavy chain        genes of at least two different specificities into murine        myeloma cells or other eukaryotic expression systems and        isolation of the polyvalent multispecific antibodies.

It is also the object of the present invention to provide an antibody bymeans of which undesired immune responses, such as a HAMA response, canbe avoided.

Thus, in a preferred embodiment the multivalent multimeric antibody ofthe present invention is devoid of constant regions. This is, e.g.,achieved by constructing a recombinant molecule, which consists only ofnon-immunogenic immunoglobulin variable (V_(H) and V_(L)) domains. Thiscan also be achieved by using the antibody domains of human origin. Suchantibodies can be prepared by methods known to the person skilled in theart, e.g. by the following methods:

-   -   (a) Construction of the multivalent multispecific single chain        Fv-antibodies by combining the genes encoding at least four        immunoglobulin variable V_(H) and V_(L) domains, either        separated by peptide linkers or by no linkers, into a single        genetic construct and expressing it in bacteria or other        appropriate expression system.    -   (b) Non-covalent heterodimerization of two hybrid scFv        fragments, each consisting of V_(H) and V_(L) domains of        different specificity (against CD19 or CD16), either separated        by peptide linkers or by no linkers, as a result of        co-expression of corresponding genes or co-refolding of        separately expressed corresponding precursors.    -   (c) Non-covalent homodimerization of single chain Fv-antibodies        comprising at least four V_(H) and V_(L) domains of different        specificity (against CD19 or CD16) either separated by peptide        linkers or by no linkers, in an orientation preventing their        intramolecular pairing.

In a preferred embodiment, the multivalent multimeric antibody of thepresent invention is a single chain Fv-antibody comprising at least fourimmunoglobulin variable V_(H) and V_(L) domains, either separated bypeptide linkers or by no linkers.

The term “Fv-antibody” as used herein relates to an antibody containingvariable domains but not constant domains. The term “peptide linker” asused herein relates to any peptide capable of connecting two variabledomains with its length depending on the kinds of variable domains to beconnected. The peptide linker might contain any amino acid residue withthe amino acid residues glycine, serine and proline being preferred.

In a more preferred embodiment, the multivalent multimeric antibody ofthe present invention is a single chain Fv-antibody comprising at leastfour immunoglobulin variable V_(H) and V_(L) domains, either separatedby peptide linkers or by no linkers. Such an antibody can be generated,e.g., by combining the genes encoding at least four immunoglobulinvariable V_(H) and V_(L) domains, either separated by peptide linkers orby no linkers, into a single genetic construct and expressing it inbacteria or other appropriate expression system.

In a further more preferred embodiment, the multivalent multimericantibody of the present invention is a heterodimer of two hybrid singlechain Fv-antibodies, each consisting of V_(H) and V_(L) domains ofdifferent specificity against CD19 or CD16, either separated by peptidelinkers or by no linkers. Such an antibody can be generated, e.g., bynon-covalent heterodimerization of two hybrid single chainFv-antibodies, each consisting of V_(H) and V_(L) domains of differentspecificity (against CD19 or CD16), either separated by peptide linkersor by no linkers, as a result of co-expression of corresponding genes orco-refolding of separately expressed corresponding precursors.

In an alternative embodiment, the multivalent multimeric antibody of thepresent invention is a homodimer of single chain Fv-antibodiescomprising at least four V_(H) and V_(L) domains of differentspecificity against CD19 or CD16, either separated by peptide linkers orby no linkers.

The present invention also relates to a multivalent multimeric antibody,wherein said antigen-binding domains mimic or correspond to V_(H) andV_(L) regions from a natural antibody. Preferably, said natural antibodyis a monoclonal antibody, synthetic antibody, or humanized antibody.

In a further preferred embodiment, the multivalent multimeric antibodyof the present invention is an antibody, wherein (a) the first hybridsingle chain Fv-antibody is V_(H)16-V_(L)19 and the second hybrid singlechain Fv-antibody is V_(H)19-V_(L)16; or (b) the first hybrid singlechain Fv-antibody is V_(L)16-V_(H)19 and the second hybrid single chainFv-antibody is V_(L)19-V_(H)16.

Preferably, the peptide linker connecting the variable V_(H) and V_(L)domains comprises comprises 0 to 12 amino acids, preferably 10 aminoacids.

In a further preferred embodiment, the variable V_(H) or V_(L) domainsof the multivalent multimeric antibody are shortened by at least oneamino acid residue at their N— and/or C-terminus. In a more preferredembodiment, the multivalent multimeric antibody of the present inventionis an antibody wherein (a) the first hybrid single chain Fv-antibody isV_(H)16-V_(L)19 and the second hybrid single chain Fv-antibody isV_(H)19-V_(L)16; or (b) the first hybrid single chain Fv-antibody isV_(L)16-V_(H)19 and the second hybrid single chain Fv-antibody isV_(L)19-V_(H)16 and wherein the two hybrid single chain Fv-antibodiesare connected via a peptide linker. A preferred length of this peptidelinker is 0-30 amino acids with a length of 12 amino acid being morepreferred.

The peptide linkers of the multivalent multimeric antibodies of thepresent invention might comprise any amino acid residue, howeveralanine, glycine, serine and proline residues are preferred.

The non-covalent binding of the multivalent multimeric antibody of thepresent invention can be strengthened by the introduction of at leastone disulfide bridge between at least one pair of V-domains. This can beachieved by modifying the DNA sequences encoding the variable domainsaccordingly, i.e. by inserting in each of the DNA sequences encoding thetwo domains a codon encoding a cysteine residue or by replacing a codonby a codon encoding a cysteine residue.

Finally, the multivalent multimeric antibody of the present inventioncan be further modified using conventional techniques known in the art,for example, by using amino acid deletion(s), insertion(s),substitution(s), addition(s), and/or recombination(s) and/or any othermodification(s) known in the art either alone or in combination. Methodsfor introducing such modifications in the DNA sequence underlying theamino acid sequence of a variable domain or peptide linker are wellknown to the person skilled in the art; see, e.g., Sambrook, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.

The multivalent multimeric antibodies of the present invention cancomprise at least one further compound, e.g., a protein domain, saidprotein domain being linked by covalent or non-covalent bonds. Thelinkage can be based on genetic fusion according to the methods known inthe art and described above or can be performed by, e.g., chemicalcross-linking as described in, e.g., WO 94/04686. The additional domainpresent in the fusion protein comprising the antibody employed inaccordance with the invention may preferably be linked by a flexiblelinker, advantageously a peptide linker, wherein said peptide linkercomprises plural, hydrophilic, peptide-bonded amino acids of a lengthsufficient to span the distance between the C-terminal end of saidfurther protein domain and the N-terminal end of the antibody or viceversa. The above described fusion protein may further comprise acleavable linker or cleavage site for proteinases. Thus, e.g., at leastone monomer of the antibody might be linked to a an effector moleculehaving a conformation suitable for biological actvity or selectivebinding to a solid support, a biologically active substance (e.g. acytokine or growth hormone), a chemical agent (e.g. doxorubicin,cyclosporin), a peptide (e.g. α-amanitin), a protein (e.g. granzyme Aand B) or a drug.

Another object of the present invention is a process for the preparationof a multivalent multimeric Fv-antibody according to the presentinvention, wherein (a) DNA sequences encoding the peptid linkers areligated with the DNA sequences encoding the variable domains such thatthe peptide linkers connect the variable domains resulting in theformation of a DNA sequence encoding a monomer of the multivalentmultimeric Fv-antibody and (b) the DNA sequences encoding the variousmonomers are expressed in a suitable expression system. The varioussteps of this process can be carried according to standard methods, e.g.methods described in Sambrook et al., or described in the Examples,below.

The present invention also relates to a polynucleotide encoding themultivalent multimeric antibody of the present invention and vectors,preferably expression vectors containing said polynucleotides.

A variety of expression vector/host systems may be utilized to containand express sequences encoding the multivalent multimeric antibody.These include, but are not limited to, microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid, or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids);or animal cell systems. The invention is not limited by the host cellemployed.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector-enhancers, promoters, 5′ and 3′untranslated regions which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript.RTM. phagemid (Stratagene,LaJolla, Calif.) or pSport1.TM. plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding themultivalent multimeric antibody, vectors based on SV40 or EBV may beused with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the multivalent multimeric antibody.Vectors suitable for use in the present invention include, but are notlimited to the pSKK expression vector for expression in bacteria.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Grant et al. (1987)Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding the multivalent multimeric antibody may be driven byany of a number of promoters. For example, viral promoters such as the35S and 19S promoters of CaMV may be used alone or in combination withthe omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.6:307-311). Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. and Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

An insect system may also be used to express the multivalent multimericantibody of the present invention. For example, in one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes in Spodoptera frugiperda cells or inTrichoplusia larvae. The sequences encoding the multivalent multimericantibody may be cloned into a non-essential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the gene encoding the multivalentmultimeric antibody will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which APOP may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding the multivalent multimeric antibody may beligated into an adenovirus transcription/translation complex consistingof the late promoter and tripartite leader sequence. Insertion in anon-essential E1 or E3 region of the viral genome may be used to obtaina viable virus which is capable of expressing the multivalent multimericantibody in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding the multivalent multimeric antibody.Such signals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the multivalent multimeric antibody, itsinitiation codon, and upstream sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in case where onlycoding sequence is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed antibody chains in the desired fashion. Post-translationalprocessing which cleaves a “prepro” form of the protein may also be usedto facilitate correct insertion, folding and/or function. Different hostcells which have specific cellular machinery and characteristicmechanisms for post-translational activities (e.g., CHO, HeLa, MDCK,HEK293, and W138), are available from the American Type CultureCollection (ATCC; Bethesda, Md.) and may be chosen to ensure the correctmodification and processing of the foreign antibody chains.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cell lines which stably expressthe multivalent multimeric antibody may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, beta-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

A particular preferred expression vector is pSKID19×16 deposited withthe DSMZ (Deutsche Sammlung fur Mikroorganismen und Zellen) according tothe Budapest Treaty under DSM 14529 on Sep. 24, 2001.

The present invention also relates to a composition containing amultivalent multimeric antibody of the present invention, apolynucleotide or an expression vector. Preferably, said composition isa pharmaceutical composition preferably combined with a suitablepharmaceutical carrier or a diagnostic composition optionally furthercomprising suitable means for detection. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g. byintravenous, intraperetoneal, subcutaneous, intramuscular, topical orintradermal administration. The route of administration, of course,depends on the nature of the disease, e.g. tumor, and the kind ofcompound contained in the pharmaceutical composition. The dosage regimenwill be determined by the attending physician and other clinicalfactors. As is well known in the medical arts, dosages for any onepatient depends on many factors, including the patient's size, bodysurface area, age, sex, the particular compound to be administered, timeand route of administration, the kind of the disorder, general healthand other drugs being administered concurrently.

Preferred medical uses of the compounds of the present inventiondescribed above are the treatment of a B-cell malignancy, preferablynon-Hodgkin disease, a B-cell mediated autoimmune disease or a depletionof B-cells.

Another subject matter of the present invention relates to a diagnostickit, comprising: (a) a multivalent multimeric antibody according to theinvention, (b) an expression vector according to the invention, and,optionally, (c) conventional auxiliary agents, such as buffers, solventsand controls. The multivalent multimeric antibody can be detectablylabeled. In a preferred embodiment, said kit allows diagnosis by ELISAand contains the antibody bound to a solid support, for example, apolystyrene microtiter dish or nitrocellulose paper, using techniquesknown in the art. Alternatively, said kit is based on a RIA and containssaid antibody marked with a radioactive isotope. In a preferredembodiment of the kit of the invention the multivalent multimericantibody is labelled with enzymes, fluorescent compounds, luminescentcompounds, ferromagnetic probes or radioactive compounds.

The below examples explain the invention in more detail.

EXAMPLE 1 Construction of the Plasmids pKID19×16 and pSKID19×16 for theExpression of Bivalent Bispecific CD19×CD16 Diabody in Bacteria

The genes coding for V_(H)16-V_(L)19 and V_(H)19-V_(L)16 hybrid scFvswere constructed by exchange of the anti-CD3 V_(H) and V_(L) genes inplasmids pHOG3-19 and pHOG19-3 (Kipriyanov et al., 1998, Int. J. Cancer77, 763-772) for their anti-human CD16 counterparts (Arndt et al., 1999,Blood 94 2562-2568) using NcoI/HindIII and HindIII/XbaI restrictionsites, respectively. The expression plasmid pKID19×16 containingdicistronic operon for cosecretion of two hybrid scFv was constructed byligation of the BglII/XbaI restriction fragment from pHOG16-19comprising the vector backbone and the BglII/XbaI fragment frompHOG19-16 (FIG. 3).

To increase the yield of functional CD19×CD16 BsDb in the bacterialperiplasm, an optimized expression vector pSKID19×16 was generated (FIG.4). This vector was constructed on the base of plasmid pHKK (Horn etal., 1996, Appl. Microbiol. Biotechnol. 46, 524-532) containing hok/sokplasmid-free cell suicide system (Thisted et al., 1994, EMBO J. 13,1960-1968). First, the gene coding for hybrid scFv V_(H)3-V_(L)19 wasamplified by PCR from the plasmid pHOG3-19 (Kipriyanov et al., 1998,Int. J. Cancer 77, 763-772) using the primers 5-NDE,5′-GATATACATATGAAATACCTATTGCCTACGGC-3′, and 3-AFL,5′-CGAATTCTTAAGTTAGCACAGGCCTCTAGAGACACACAGATCTTTAG-3′. The resulting 921bp PCR fragment was digested with NdeI and AflII and cloned into theNdeI/AflII linearized plasmid pHKK generating the vector pHKK3-19. Todelete an extra XbaI site, a fragment of pHKK plasmid containing3′-terminal part of the lacI gene (encodes the lac repressor), thestrong transcriptional terminator t_(HP) and wild-type lacpromoter/operator was amplified by PCR using primers 5-NAR,5′-CACCCTGGCGCCCAATACGCAAACCGCC-3′, and 3-NDE,5′-GGTATTTCATATGTATATCTCCTTCTTCAGAAATTCGTAATCATGG-3′. The resulting 329bp DNA fragment was digested with NarI and NdeI and cloned intoNarI/NdeI linearized plasmid pHKK3-19 generating the vector pHKKDXba. Tointroduce a gene encoding the Skp/OmpH periplasmic factor for higherrecombinant antibody production (Bothmann and Plückthun, 1998, Nat.Biotechnol. 16, 376-380), the skp gene was amplified by PCR with primersskp-3, 5′-CGAATTCTTAAGAAGGAGATATACATATGAAAAAGTGGTTATTAGCTGCAGG-3′ andskp-4, 5′-CGAATTCTCGAGCATTATTTAACCTGTTTCAGTACGTCGG-3′ using as atemplate the plasmid pGAH317 (Holck and Kleppe, 1988, Gene 67, 117-124).The resulting 528 bp PCR fragment was digested with AflII and XhoI andcloned into the AflII/XhoI digested plasmid pHKKDXba resulting in theexpression plasmid pSKK2.

The gene encoding the hybrid scFv V_(H)16-V_(L)19 was isolated as 820 bpDNA fragment after digestion of pHOG16-19 with NcoI and XbaI. This genewas cloned into NcoI/XbaI linearized vector pSKK2 resulting in plasmidpSKK16-19. The gene encoding the second hybrid scFv V_(H)19-V_(L)16 wasamplified from the plasmid pHOG19-16 by PCR with the primers 5-BGL,5′-GCACACAGATCTGAGAAGGAGATATACATATGAAATACCTATTGCCTACGGC-3′, and pSEXBn,5′-GGTCGACGTTAACCGACAAACAACAGATAAAACG-3′. The resulting PCR fragment wasdigested with BglII and XbaI and cloned into the BglII/XbaI linearizedplasmid pSKK16-19 generating the expression vector pSKID19×16 (FIG. 4).This vector contains several features that improve plasmid performanceand lead to increased accumulation of functional bivalent product in theE. coli periplasm under conditions of both shake-flask cultivation andhigh cell density fermentation. These are the hok/sok post-segregationkilling system, which prevents plasmid loss, strong tandemribosome-binding sites and a gene encoding the periplasmic factorSkp/OmpH that increases the functional yield of antibody fragments inbacteria. The expression cassette is under the transcriptional controlof the wt lac promoter/operator system and includes a short sequencecoding for the N-terminal peptide of beta-galactosidase (laczí) with afirst rbs derived from the E. coli lacZ gene, followed by genes encodingthe two hybrid scFvs and Skp/OmpH periplasmic factor under thetranslational control of strong rbs from gene 10 of phage T7 (T7g10).

The nucleotide and protein sequences of the CD19×CD16 BsDb in plasmidpSKID19×16 are indicated in FIG. 5.

EXAMPLE 2 Production in Bacteria and Purification of Bivalent BispecificCD19×CD16 Diabody

The E. coli XL1-Blue cells (Stratagene, La Jolla, Calif.) transformedwith the expression plasmid pKID19×16 or E. coli strain RV308 (Maurer etal., 1980, J. Mol. Biol. 139, 147-161) transformed with the expressionplasmid pSKID19×16 were grown overnight in 2×YT medium with 50 μg/mlampicillin and 100 mM glucose (2×YT_(GA)) at 37° C. or 26° C.,respectively. Dilutions (1:50) of the overnight cultures in 2×YT_(GA)were grown as flask cultures at 37° C. (XL1-Blue) or at 26° C. (RV308)with vigorous shaking (180-220 rpm) until optical density (OD) 600 nmreached 0.8-0.9. Bacteria were harvested by centrifugation at 5,000 gfor 10 min at 20° C. and resuspended in the same volume of fresh YTBSmedium (2×YT containing 1 M sorbitol, 2.5 mM glycine betaine and 50μg/ml ampicillin. Isopropyl-β-D-thiogalactopyranoside (IPTG) was addedto a final concentration of 0.2 mM and growth was continued at 20° C.for 16-18 h. Cells were harvested by centrifugation at 9,000 g for 20min and 4° C. In case of using RV308/pSKID19×16, the culture supernatantwas discarded. In contrast, the supernatant was retained and kept on icefor XL1-Blue/pKID19×16. To isolate soluble periplasmic proteins, thepelleted bacteria were resuspended in 5% of the initial volume ofice-cold 50 mM Tris-HCl, 20% sucrose, 1 mM EDTA, pH 8.0. After 1 hincubation on ice with occasional stirring, the spheroplasts werecentrifuged at 30,000 g for 30 min and 4° C. leaving the solubleperiplasmic extract as the supernatant and spheroplasts plus theinsoluble periplasmic material as the pellet. For RV308/pSKID19×16,periplasmic extract was thoroughly dialyzed against 50 mM Tris-HCl, 1 MNaCl, pH 7.0, and used as a starting material for isolating CD19×CD16BsDb. For XL1-Blue/pKID19×16, the culture supernatant and the solubleperiplasmic extract were combined and clarified by additionalcentrifugation (30,000 g, 4° C., 40 min). The recombinant product wasconcentrated by ammonium sulfate precipitation (final concentration 70%of saturation). The protein precipitate was collected by centrifugation(10,000 g, 4° C., 40 min) and dissolved in 10% of the initial volume of50 mM Tris-HCl, 1 M NaCl, pH 7.0 (starting material). Immobilized metalaffinity chromatography (IMAC) was performed at 4° C. using a 5 mlcolumn of Chelating Sepharose (Amersham Pharmacia, Freiburg, Germany)charged with cu²⁺ and equilibrated with 50 mM Tris-HCl, 1 M NaCl, pH 7.0(start buffer). The sample was loaded by passing the sample over thecolumn. It was then washed with twenty column volumes of start bufferfollowed by start buffer containing 50 mM imidazole until the absorbance(280 nm) of the effluent was minimal (about thirty column volumes).Absorbed material was eluted with 50 mM Tris-HCl, 1 M NaCl, 250 mMimidazole, pH 7.0. The final purification was achieved by ion-exchangechromatography on a Mono Q HR 5/5 column (Amersham Pharmacia) in 20 mMTris-HCl, pH 8.5 with a linear 0-1 M NaCl gradient. The fractionscontaining diabody were concentrated with simultaneous buffer exchangefor PBS containing 50 mM imidazole, pH 7.0 using Ultrafree-15centrifugal filter device (Millipore, Eschborn, Germany). Proteinconcentrations were determined by the Bradford dye-binding assay(Bradford, 1976, Anal. Biochem., 72, 248-254) using the Bio-Rad (Munich,Germany) protein assay kit. Purified CD19×CD16 BsDb was mainly in adimeric form with a molecular mass (M_(r)) around 60 kDa, asdemonstrated by gel filtration on a calibrated Superdex 200 HR 10/30column (Amersham Pharmacia) (FIG. 6). SDS-PAGE analysis demonstratedthat the BsDb could be resolved into two protein bands corresponding tothe calculated M_(r) of 28,730 for V_(H)16-V_(L)19 scFv and M_(r) of29,460 for V_(H)19-V_(L)16 scFv (FIG. 7).

EXAMPLE 3 Determination of Diabody Affinity by Surface Plasmon Resonance(SPR)

Kinetic constants of interaction of CD19×CD16 BsDb with extracellulardomain (ECD) of human CD16 were determined by SPR using BIAcore 2000biosensor system (Biacore, Uppsala, Sweden). For immobilization on astreptavidin coated sensor chip SA (Biacore), the CD16 ECD wasbiotinylated according to a modified protocol of the ECL proteinbiotinylation module (Amersham Pharmacia). As a negative control,biotinylated porcine tubulin was used. The biotinylated antigens dilutedin HBS-EP buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005%polyoxyethylenesorbitan; Biacore) at a concentration of 10 μg/ml wereapplied to a sensor chip at a flow rate of 5 μl/min for 4 min resultingin immobilization of 800 resonance units (RU) of CD16 ECD and 900 RU oftubulin. All SPR measurements were carried out at a flow rate of 20μl/min in HBS-EP at 25° C. Analyses were performed at eight diabodyconcentrations from 6.25 to 800 nM. Each injected sample (100 μl) was incontact with immobilized antigen for 5 min. The dissociation wasfollowed for 10 min. After each cycle, the surface of the sensor chipwas flushed with the buffer. Kinetic constants were calculated accordingto 1:1 (Langmuir) binding model using BIAevaluation version 3.0 software(Biacore). CD19×CD16 BsDb exhibited a fairly high off-rate from CD16coated sensor chip, thus making the regeneration of the biosensorsurface unnecessary. The calculated off- and on-rate constants were2.3×10⁻² s⁻¹ and 2.7×10⁴ s⁻¹ M⁻¹, respectively, resulting in a K_(d) of8.5×10⁻⁷ M (Table 1). A nearly identical affinity constant was deducedfrom the evaluation of steady-state binding levels (Table 1). TABLE 1Affinity and kinetics of BsDb CD19 × CD16 binding to CD16 ECD asdetermined by SPR k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) t_(1/2)(min) K_(d) (M)K_(eq) (M) 2.7 × 10⁴ 2.3 × 10⁻² 0.5 8.5 × 10⁻⁷ 8.7 × 10⁻⁷k_(off) and k_(on) values were measured by SPR using immobilizedbiotinylated CD16 ECD. Affinity constants were calculated either fromthe ratio k_(off)/k_(on) (K_(d)) or from the steady-state analysis ofSPR data (K_(eq)). The half-life (t_(1/2)) for dissociation of# diabody-antigen complex was deduced from the ratio ln2/k_(off).

EXAMPLE 4 Cell Binding Measurements

The human CD19⁺ B cell line JOK-1 and the human embryonic kidney (HEK)293 cells stably transfected with human CD16B cDNA (293-CD16) cells wereused for flow cytometry experiments. In brief, 5×10⁵ cells in 50 μl RPMI1640 medium (GIBCO BRL, Eggestein, Germany) supplemented with 10% FCSand 0.1% sodium azide (referred to as complete medium) were incubatedwith 100 ml of diabody preparation for 45 min on ice. After washing withcomplete medium, the cells were incubated with 100 μl of 10 μg/mlanti-c-myc MAb 9E10 in the same buffer for 45 min on ice. After a secondwashing cycle, the cells were incubated with 100 μl of FITC-labeled goatanti-mouse IgG (GIBCO BRL) under the same conditions as before. Thecells were then washed again and resuspended in 100 μl of 1 μg/mlsolution of propidium iodide (Sigma, Deisenhofen, Germany) in completemedium to exclude dead cells. Fluorescence of stained cells was measuredusing a FACScan flow cytometer (Becton Dickinson, Mountain View,Calif.). Mean fluorescence (F) was calculated using Cellquest software(Becton Dickinson) and the background fluorescence was subtracted.Equilibrium dissociation constants (K_(eq)) were determined by fittingthe experimental values to the Lineweaver-Burk equation:1/F=1/F_(max)+(K_(eq)/F_(max))(1/[BsDb]) using the software programGraphPad Prism (GraphPad Software, San Diego, Calif.). The flowcytometry experiments demonstrated a specific interaction of CD19×CD16BsDb with both CD19⁺ JOK-1 cells and 293-CD16 cells expressing ECD ofhuman CD16 on their surface. However, the fluorescence intensitiesobtained for interaction with JOK-1 cells were significantly higher thanfor 293-CD16 cells reflecting the 6-fold difference in affinity valuesfor the two antigen-binding sites (FIG. 8, Table 2).

EXAMPLE 5 in vitro Cell Surface Retention

To investigate the biological relevance of the differences in directbinding experiments, the in vitro retention of the CD19×CD16 BsDb on thesurface of both CD19⁺ and CD16⁺ cells at 37° C. was determined by flowcytometry (FIG. 9). Cell surface retention assays were performed at 37°C. under conditions preventing internalization of cell surface antigens,as described (Adams et al., 1998, Cancer Res. 58, 485-490), except thatthe detection of retained diabody was performed using anti-c-myc MAb9E10 (IC Chemikalien) followed by FITC-labeled anti-mouse IgG (GIBCOBRL). Kinetic dissociation constant (k_(off)) and half-life (t_(1/2))values for dissociation of diabody were deduced from a one-phaseexponential decay fit of experimental data using GraphPad Prism(GraphPad Software). CD19×CD16 BsDb had a relatively short retentionhalf-life (t_(1/2)) on 293-CD16 cells (3.6 min) and 3-fold longert_(1/2) on the surface of CD19⁺ JOK-1 cells, thus correlating well withthe lower CD16 binding affinity deduced from direct binding experiments(Table 2). To determine whether the CD19 activity of diabody isinfluenced by the second moiety of the bispecific molecule, CD19×CD3BsDb (Cochlovius et al., 2000, J. Immunol. 165, 888-895; Kipriyanov etal., 1998, Int. J. Cancer 77, 763-772) was used as a control in all flowcytometry experiments. Direct binding and cell surface retention onCD19⁺ JOK-1 cells were practically indistinguishable for both diabodies.The calculated K_(eq) and t_(1/2) values were 5.7 nM and 10.8 min,respectively, for CD19×CD3 BsDb, and 6.1 nM and 10.6 min for CD19×CD16BsDb. These results indicate that the second specificity present in thebispecific diabody does not significantly affect the affinity forbinding to CD19. TABLE 2 Affinity and kinetics of BsDb CD19 × CD16binding to cell surface antigens Cells k_(off) (s⁻¹) t_(1/2)(min) K_(eq)(M) JOK-1 (CD19⁺) 1.1 × 10⁻³ 10.6 6.1 × 10⁻⁹ 293-CD16 (CD16⁺) 3.2 × 10⁻³ 3.6 3.9 × 10⁻⁸k_(off) values were deduced from JOK-1 and 293-CD16 cell surfaceretention experiments. Equilibrium dissociation of constants (K_(eq))were deduced from Lineweaver-Burk plots shown on FIG. 8. The half-lifevalues (t_(1/2)) for dissociation of diabody-antigen complexes werededuced# from the ratio ln2/k_(off).

EXAMPLE 6 Killing Tumor Cells in vitro

The efficacy of the CD19×CD16 BsDb in mediating tumor cell lysis byhuman peripheral blood lymphocytes (PBL) or NK cells was determinedusing the JAM test, which is based on measuring DNA fragmentation in thetarget cell as a result of apoptosis (Matzinger et al., 1991, J.Immunol. Meth. 145, 185-192). The CD19-expressing Burkitt's lymphomacell line Raji was used as target cells. As effector cells, eitheractivated human PBLs prepared as described (Kirpiyanov et al., 1998,Int. J. Cancer 77, 763-772), or the human NK cells were used. NK cellswere isolated from peripheral blood by magnetic cell sorting using theNK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Forthe cell kill assay, 10⁵ effector cells were mixed in round-bottomedmicrotiter plates with 10⁴ target cells labeled with [³H]-thymidine in100 μl medium plus 50 μl of diabody sample. After incubating the plateat 37° C., 5% CO₂ for 4 h, the cells were harvested and radioactivitywas measured with a liquid scintillation beta counter (LKB, Wallach,Germany). Cytotoxicity related to the apoptosis-induced DNAfragmentation was calculated as % specific killing=(S-E)/S×100, where Eis experimentally retained labeled DNA in the presence of killers (incpm) and S is retained DNA in the absence of killers (spontaneous).Difference of means was evaluated by a paired t-test using GraphPadPrism (GraphPad Software). The death of CD19⁺ Raji cells in the presenceof freshly prepared PBL from a healthy donor was specifically triggeredby CD19×CD16 BsDb in a dose-dependent manner resulting in 45% ofspecific killing at a BsDb concentration of 5 μg/ml and E:T ratio of50:1 (FIG. 10). Substitution of PBL by NK cells isolated from the bloodof the same donor further increased the cytotoxic effect of CD19×CD16BsDb up to 60% under the same conditions (FIG. 11).

EXAMPLE 7 Treatment of Burkittís Lymphoma in Severe CombinedImmunodeficiency (SCID) Mice

The SCID mice were obtained from Charles River (Sulzfeld, Germany) andkept under specific pathogen-free conditions at the Central AnimalFacilities of the German Cancer Research Center. In each experiment,cohorts of five animals were used to permit accurate comparisons amongdifferently treated groups. Mice were irradiated (300 rad) and receivedi.p. injections of 10 μl of anti-asialo-GM1 monoclonal antibody (Wako,Neuss, Germany) according to the manufacturerís suggestions. One daylater, 107 Raji cells were injected s.c. dorsolaterally leading to thelocally growing tumors. Treatment was started after the tumors reached asize of 5 mm in diameter (day 0). At days 0, 7, and 15, the animalsreceived i.v. injections of either PBS (control group) or 5×10⁶ humanPBLs. Four h after each PBL inoculation, the mice were treated eitherwith phosphate buffered saline (PBS) or with 50 μg CD19×CD16 BsDbadministered via the tail vein. Tumor size was measured using a caliperevery 2^(nd) day. Animals were followed until the s.c. tumors reached amaximal tolerated size of 15 mm in diameter and were killed by cervicaldislocation. The days of sacrifice were recorded and were used forsurvival time analysis. The surviving animals were followed up to 60days after the first treatment. For statistical evaluation, thefollow-up duration of the tumor-treatment experiment was 30 days (end ofexperiment). The median survival times were estimated by the methoddescribed by Kaplan and Meier (1958, J. Am. Statist. Assoc. 53,457-481). Differences between survival curves were compared using alogrank test (Mantel and Haenszel, 1959, J. Natl. Cancer Inst. 22,719-748).

All animals in the control groups receiving PBS or PBL alone did notshow any tumor suppression and developed tumors larger than 1.5 cm indiameter in less than 3 weeks (FIG. 12). There was no significantdifference between tumor growth in mice receiving PBS and mice receivingactivated PBLs alone, which indicated that under the conditions used,any allogeneic reaction of the effector cells towards the tumor could beignored. The animals were sacrificed when the tumors reached the maximumtolerated size of 15 mm in diameter. Sacrifice dates were recorded, andthe median survival was calculated for each group (FIG. 13). The mediansurvival times were not significantly different in the control groupsreceiving PBS and human PBLs alone at 21.5 and 23 days, respectively(P=0.4469). In contrast to control groups, all mice receiving aCD19×CD16 BsDb demonstrated significant tumor regression. The animalsreceiving three injections of diabody displayed a minimal tumor size ondays 15-20, when 2 out of 5 mice were tumor-free. Afterwards, however,the tumors started to grow again with comparable rates in all animals ofthis group (FIG. 12). The median survival time calculated for the groupreceiving CD19×CD16 BsDb (32.5 days) was significantly different fromthe control groups (P<0.01). These in vivo data clearly confirms thestrong antitumor effect of CD19×CD16 BsDb, which recruits human NK cellsto the tumor target.

1. A multivalent multimeric antibody, characterized by the followingfeatures: (a) it has at least two specificities; (b) at least oneantigen-binding domain is specific to human CD19; and (c) at least oneantigen-binding domain is specific to human CD
 16. 2. The multivalentmultimeric antibody according to claim 1, wherein CD19 antigen isexpressed on human B cells.
 3. The multivalent multimeric antibodyaccording to claim 2, wherein CD16 antigen is expressed on human NKcells.
 4. The multivalent multimeric antibody according to claim 1,which is devoid of constant regions.
 5. The multivalent multimericantibody according to claim 4, which is a single chain Fv-antibodycomprising at least four immunoglobulin variable V_(H) and V_(L)domains, either separated by peptide linkers or by no linkers.
 6. Themultivalent multimeric antibody according to claim 4, which is aheterodimer of two hybrid single chain Fv-antibodies, each consisting ofV_(H) and V_(L) domains of different specificity against cD19 or CD16,either separated by peptide linkers or by no linkers.
 7. The multivalentmultimeric antibody according to claim 4, which is a homodimer of singlechain Fv-antibodies comprising at least four V_(H) and V_(L) domains ofdifferent specificity against CD19 or CD16, either separated by peptidelinkers or by no linkers.
 8. The multivalent multimeric antibodyaccording to claim 1, wherein said antigen-binding domains mimic orcorrespond to V_(H) and V_(L) regions from a natural antibody.
 9. Themultivalent multimeric antibody according to claim 8, wherein saidnatural antibody is a monoclonal antibody, synthetic antibody, orhumanized antibody.
 10. The multivalent multimeric antibody according toclaim 6, wherein (a) the first hybrid single chain Fv-antibody isV_(H)16-V_(L)19 and the second hybrid single chain Fv-antibody isV_(H)19-V_(L)16; or (b) the first hybrid single chain Fv-antibody isV_(L)16-V_(H)19 and the second hybrid single chain Fv-antibody isV_(L)19-V_(H)16.
 11. The multivalent multimeric antibody according toclaim 6, comprising a peptide linker.
 12. The multivalent multimericantibody according to claim 11, wherein said peptide linker comprises 10amino acids.
 13. The multivalent multimeric antibody to claim 6, whereinthe variable V_(H) or V_(L) domains are shortened by at least one aminoacid residue at their N- and/or C-terminus.
 14. The multivalentmultimeric antibody according to claim 10, wherein two hybrid singlechain Fv-antibodies are connected via a peptide linker.
 15. Themultivalent multimeric antibody according to claim 14, wherein saidpeptide linker has a length of 10-30 amino acids.
 16. The multivalentmultimeric antibody according to claim 15, wherein said peptide linkerhas a length of 12 amino acids.
 17. The multivalent multimeric antibodyaccording to claim 16, wherein said peptide linkers comprise alanine,glycine, serine and proline residues.
 18. The multivalent multimericantibody according to claim 6, wherein the non-covalent binding of atleast one pair of V-domains is strengthened by at least one disulfidebridge.
 19. The multivalent multimeric antibody according to claim 6 anyone of claims 6 to 18, wherein at least one monomer is linked to aneffector molecule having a conformation suitable for biological activityor selective binding to a solid support, a biologically activesubstance, a chemical agent (a peptide, a protein or a drug.
 20. Apolynucleotide, which encodes a multivalent multimeric antibody of claim6.
 21. An expression vector comprising the polynucleotide of claim 20.22. The expression vector of claim 21, which is pSKID19×16 (DSM14529).23. A host cell containing the expression vector of claim
 21. 24. Aprocess for the preparation of a multivalent multimeric antibodyaccording to claim 11, wherein (a) DNA sequences encoding the peptidelinkers are ligated with the DNA sequences encoding the variable domainssuch that the peptide linkers connect the variable domains resulting inthe formation of a DNA sequence encoding a monomer of the multivalentmultimeric antibody and (b) the DNA sequences encoding the variousmonomers are expressed in a suitable expression system.
 25. Acomposition containing the multivalent multimeric antibody of claim 11,the polynucleotide of claim 20 or the expression vector of claim
 21. 26.The composition of claim 25, which is a pharmaceutical compositionoptionally further comprising a pharmaceutically acceptable carrier or adiagnostic composition optionally further comprising suitable means fordetection.
 27. A method for treating of B-cell malignancies, B-cellmediated autoimmune diseases or the depletion of B-cells comprisingadministering a pharmaceutical composition comprising the multivalentmultimeric antibody of claim 1, the polynucleotide of claim 20 or theexpression vector of claim 21 in an amount effective to treat B-cellmalignancies, B-cell mediated autoimmune diseases or the depletion ofB-cells.
 28. The method of claim 27, wherein said B-cell malignancy isnon-Hodgkin lymphoma.
 29. A method of gene therapy the method comprisingadministering a composition comprising the polynucleotide of claim 20 orthe expression vector of claim
 21. 30. A Diagnostic kit, comprising: (a)a multivalent multimeric antibody according to claim 1; and/or (b) anexpression vector according to claim 21.